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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

ANTIBODIES: ELISA

PHAGE ENZYME LINKED IMMUNOSORBANT ASSAY (ELISA)

Phage Enzyme Linked Immunosorbant Assay (ELISA)
Contributor: The Laboratories of Andrew Bradbury at Los Alamos National Laboratory and James D. Marks University of California, San Francisco
 
Overview
This protocol describes the detection of bacteriophage-derived antibodies specific for an antigen of interest. The antigen is adsorbed on the plastic wells of a 96-well microtiter plate. Enzyme Linked Immunosorbant Assay (ELISA), with a horseradish peroxidase-linked antibody against the phage, is used to identify the clones that produce phage antibodies with high affinity for the antigen. Since each phage particle contains multiple pVIII protein molecules, the detection of phage is amplified with anti-pVIII protein antibodies.
 
Procedure
1. Coat the wells of a 96-well ELISA microtiter plate (Nunc, Maxisorp™) overnight at 4°C with 100 μl of Antigen Solution per well (see Hint #1).

2. Discard the Antigen Solution and wash the microtiter plate wells two times with PBS.

3. Block the wells by adding 200 μl of 2% MPBS.

4. Incubate at 37°C for 2 hr.

5. Discard the MPBS Solution and wash the microtiter plate wells three times with PBS.

6. Add 50 μl of 4% MPBS to each of the microtiter plate wells.

7. Add 50 μl of culture supernatant containing phage antibodies (prepared in Protocol ID#2208).

8. Mix the solution by pipetting up and down; then incubate for 1 hr at room temperature.

9. Discard the solution and wash the microtiter plate wells three times with PBS/Tween.

10. Wash the microtiter plate wells three times with PBS.

11. Add 100 μl of Anti-Phage mAb to each microtiter plate well.

12. Incubate at room temperature for 1 hr.

13. Discard the Anti-Phage mAb and wash the microtiter plate wells three times PBS/Tween.

14. Wash the microtiter plate wells three times with PBS.

15. Add 100 μl of TMB System Solution.

16. Incubate at room temperature for 10 to 30 min in the dark.

17. A blue hue should develop within a couple of min.

18. Quench the reacton by adding 50 to 100 μl of Stop Solution.

19. Determine the absorbance at a wavelength of 450 nm (A450) with a spectrophotometer and 96-well plate reader format.

Solutions
2% MPBS   Prepared in PBS
2% (w/v) Non-Fat Milk Powder (Carnation™)
4% MPBS   4% (w/v) Non-Fat Milk Powder (Carnation™)
Prepared in PBS
PBS/Tween   0.1% (v/v) Tween 20
Prepared in PBS
PBS   137 mM Sodium Chloride (NaCl)
2.7 mM Potassium Chloride (KCl)
Also see Protocol ID#2152
4.3 mM Sodium Phosphate, Dibasic (Na2HPO4
1.4 mM Potassium Phosphate, Monobasic (KH2PO4)
Anti-Phage mAb   1:5000 diluted HRP-conjugated Mouse Monoclonal Anti-Phage Antibody (Pharmacia) in 2% MPBS
Antigen Solution   Also see Hint #1
10 μg/ml to 1 mg/ml Antigen in PBS
 
BioReagents and Chemicals
Sodium Phosphate, Dibasic
Potassium Chloride
HRP-conjugated Mouse Monoclonal Anti-Phage Antibody
Sodium Chloride
Potassium Phosphate, Monobasic
Non-Fat Milk Powder
Tween 20
 
Protocol Hints
1. 10 μg/ml Protein Antigen prepared in PBS is standard. Occasionally, a higher concentration or a different binding buffer is required (e.g., Carbonate buffer). If the antigen is limiting, save the supernatant after the overnight incubation. The recovered antigen can be used in the next rounds of selection.

 
Citation and/or Web Resources
1. Phage Display: A Practical Approach. Eds. T. Clackson and H. Lowman. Oxford University Press, (2001) in press.